Suppression of GTP/T alpha-dependent activation of cGMP phosphodiesterase by ADP-ribosylation by its gamma subunit in amphibian rod photoreceptor membranes

Our previous study has shown that P gamma, the regulatory subunit of cGMP p hosphodiesterase (PDE), is ADP-ribosylated by endogenous ADP-ribosyltransfe rase when P gamma is free or complexed with the catalytic subunits of PDE i n amphibian rod photoreceptor membranes. The P gamma domain containing ADP- ribosylated arginines was shown to be involved in its interaction with T al pha, a key interaction for PDE activation, In this study, we describe a pos sible function of the P gamma ADP-ribosylation in the GTP/T alpha-dependent PDE activation. When rod membranes were preincubated with or without NAD a nd washed with a buffer containing GTP, the PDE activity of NAD-preincubate d membranes was increased by the GTP-washing only to similar to 50% of that of membranes preincubated without NAD. The P gamma release by the GTP-wash ing from these NAD-preincubated membranes was also suppressed to similar to 50% of that preincubated without NAD. Taking into consideration that simil ar to 50% of P gamma is ADP-ribosylated under these conditions, these obser vations suggest that the ADP-ribosylated P gamma, cannot interact with GTP/ T alpha. We have also shown that a soluble fraction of ROS contains an enzy me(s) to release the radioactivity of [P-32] ADP-ribosylated P gamma in con centration- and time-dependent manners, suggesting that the P gamma ADP-rib osylation is reversible. Rod ADP-ribosyltransferase solubilized from membra nes by phosphatidylinositol-specific phospholipase C was separated into two fractions by ion-exchange columns. Biochemical characterization of these t wo fractions, including measurement of the K-m for NAD and P gamma, estimat ion of their molecular masses, ADP-ribosylation of P gamma arginine mutants , effects of ADP-ribosyltransferase inhibitors on the P gamma ADP-ribosylat ion, and effects of salts and pH on the P gamma ADP-ribosylation, indicates that rod ADP-ribosyltransferase contains two isozymes, and that these two isozymes have similar properties for the P gamma ADP-ribosylation. Our obse rvations strongly suggest that the negative regulation of PDE through the r eversible P gamma ADP-ribosylation may function in the phototransduction me chanism.